The zein genes of maize encode the seed storage proteins. Previous analyses have shown that zeins consist of three distinct protein types designated alpha, beta, and gamma. The alpha zeins account for approximately 70% of the total storage protein and are encoded by a multigene family. The beta and gamma zeins, which account for approximately 10% and 20% of the zein fraction, respectively, are each encoded by only one or two genes. Zein genes are actively expressed during development of normal endosperms, but their expression is quantitatively and qualitatively inhibited in several mutant genotypes. Our preliminary studies indicate that there are differences in the relative expression of genes among zein gene families that correlate with differences in the amount of cytoplasmic mRNA. To define the mechanisms regulating the expression of zein genes during endosperm formation, we propose to characterize the DNA sequences controlling their transcription and developmental regulation. Hybridization of 32P-labeled nuclear RNA to well characterized genomic clones will establish sites of transcription, timing of transcription, and whether transcription or post-transcriptional processing play a major role in the regulation of the zein gene expression. This analysis will also determine the extent to which these reactions are affected by the mutant genotypes. To define DNA sequences regulating transcription, gene flanking sequences will be ligated to a chloramphenicol acetyltransferase (CAT) gene and transferred to plant protoplasts by electroporation. Sequences required for transcription will be monitored by assaying CAT activity in protoplast extracts. We will also analyze transcriptional regulatory sequences following transformation of zein genes into tobacco and petunia plants with a Ti-plasmid vector. In this case we will monitor mRNA and protein synthesis in seeds from regenerated plants to delineate DNA sequences controlling developmental expression. Through labeling of endosperm proteins and differential screening of cDNA libraries we will attempt to identify gene products whose expression is modified in mutants that reduce the level of zein gene expression.